Therapeutic agent for treating wounds using monocyte chemotactic and activating factor

ABSTRACT

A therapeutic agent for treating wounds having properties and actions different from those of growth factors and proteins inducing growth factors, which have strong therapeutic effect is disclosed. The therapeutic agent for treating wounds according to the present invention comprises monocyte chemotactic and activating factor or a variation thereof having a monocyte-attracting property or a derivative of said monocyte chemotactic and activating factor or said variation thereof.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for treatingwounds.

BACKGROUND ART

Several factors having chemotactic properties to monocytes are known.Among these, monocyte chemotactic and activating factor (hereinafterreferred to as "MCAF") also known as MCP-1 (monocyte chemoattractantprotein-1) or GDCF (glioma-derived monocyte chemotactic factor), is aprotein consisting of 76 amino acids, which has 4 cysteine residues. Itis known that this protein has a strong chemotactic activity tomonocytes and a strong monocyte-activating property.

Identification and gene cloning of MCAF, MCP-1 and GDCF are described inthe following references:

1) K. Matsushima et al., J. Exp. Med., 169, 1485-1490, 1989;

2) Y. Furutani et al., Biochem. Biophys. Res. Commun., 159, 249-255,1989;

3) E. A. Robinson et al., Proc. Natl. Acad. Sci. USA, 86, 1850-1854,1989; and

4) T. Yoshimura et al., FEBS Letters, 244, 487-493, 1989.

General descriptions of the protein are described in the followingreference:

5) N. Mukaida et al., Microbiol. Immunol. 36, 773-789, 1992.

MCP-1 and GDCF described in the References 3) and 4) are the samesubstance as MCAF, described in the References 1) and 2).

It is known that MCAF strongly attracts and activates monocytes, anduses exploiting immunopotentiation and anti-tumor properties aresuggested in the above-mentioned references. Further, as a generalconcept, it is known that monocytes which have gathered in a wound playa role in the natural healing of the wound. However, it is not knownthat MCAF has an activity to directly promote therapy of wounds.

It is known that several proteinous growth factors promote healing ofwounds. For example, growth factors such as fibroblast growth factor(FGF), epidermal growth factor (EGF), transforming growth factor (TGF-α,TGF-β), platelet-derived growth factor (PDGF), endothelial cell growthfactor (ECGF) and keratinocyte growth factor are expected to havetherapeutic activities for wounds (Reference 6).

6) T. A. Mastoe et al., J. Clin. Invest., 87, 694-703 (1991) .

It was recently reported in U.S. Pat. No. 5,202,118 that interleukin-1(IL-1) has a therapeutic effect for wounds.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a therapeutic agent fortreating wounds, which does not employ the growth factors or proteinsinducing growth factors, but has properties and actions totallydifferent from these substances, and which has a strong therapeuticeffect.

That is, the present invention provides a therapeutic agent for treatingwounds comprising as an effective ingredient MCAF or a variant thereofhaving a monocyte-chemotactic activity or a derivative of MCAF orsaidvariation thereof.

By the present invention, a useful therapeutic agent which promoteshealing of wounds such as traumatic ulcers caused by burn, trauma,surgery and the like, and basic disorder ulcers caused by endogenousfactors (digestive fluid) and the like was provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows tensile strengths of skin samples at 7 days after wounding;

FIG. 2 shows tensile strengths of skin samples at 14 days afterwounding;

FIG. 3 shows therapeutic effects of MCAF for wounds in comparison withthose by other growth factors; and

FIG. 4 shows therapeutic effects of MCAF for wounds.

BEST MODE FOR CARRYING OUT THE INVENTION

MCAF is a protein consisting of 76 amino acids, which attracts andactivates monocytes, as described in the above-mentioned References 1)to 5). The amino acid sequence of MCAF is shown in SEQ ID NO. 1 in theSequence Listing.

In the present invention, variants of MCAF, which substantially have theamino acid sequence of MCAF, and which attract and activate monocytescan also be used as the effective ingredient. Here, the term "variantsof MCAF" means that it substantially has the amino acid sequence ofMCAF, and the term includes those compounds in which amino acids in theamino acid sequence of MCAF are naturally or artificially deleted, addedor substituted to the extent that the monocyte-attracting property ofMCAF is not lost. Sequences in the N-terminal and C-terminal regions ofnaturally occurring and recombinant MCAF may vary (increase or decreasein amino acid residues) depending on the production conditions and theseare included in the scope of the present invention. Further, in thepresent invention, derivatives obtained by chemically or biochemicallymodifying MCAF or the above-mentioned MCAF variants (e.g., thoseobtained by chemically linking polyethylene glycol or an analoguethereof; those obtained by attaching phosphate or sulfate groups; thosetreated with a peptidase such as an endopeptidase; those treated with asugar chain-modifying enzyme or a sugar chain-attaching enzyme, such assialidase) may also be used as the effective ingredient. Analogues ofMCAF having the number of amino acids and arrangement of cysteineresidues, which are similar to those in MCAF, are known. In human,RANTES, MCP-2, MCP-3, LD78, ACT2 and I-309 are known, and in mouse, JE,MIP-1α, MIP-β, TCA-3 and the like are known as structural analogues ofMCAF (above-described References 4) and 5)). Recently, MCP-2 and MCP-3were reported as MCAF analogues (Reference 6: J. Van Damme et al., J.Exp. Med., 176, 59-65, 1992)). Among these, those which attract oractivate monocytes or macrophages are included in the variants of MCAFin the present invention and may be used as an effective ingredient ofthe therapeutic agent for treating wounds according to the presentinvention. In particular, since it has been reported that RANTES, MCP-2,MCP-3 and JE attract monocytes and macrophages (the above-mentionedReferences 5) and 6)), these substances and variants thereof, as well astheir derivatives can be used as an effective ingredient in thetherapeutic agent for treating wounds according to the presentinvention.

The process for producing MCAF is not restricted and MCAF produced byknown methods may be suitably employed. For example, purified MCAF maybe obtained by ligating the cDNA described in the above-mentionedReference 2) or 4) downstream of an appropriate regulatory region suchas the promoter of SV40 or cytomegalovirus, a promoter of baculovirus, apromoter of amino acid-synthesizing and metabolizing gene, a promoter ofsugar-synthesizing and metabolizing gene or the like to obtain anexpression vector; introducing the expression vector into animal cells,insect cells, eukaryotic unicellular organisms, prokaryotic cells or thelike to make the cells produce MCAF; and purifying the produced MCAF byappropriately combining column chromatographies such as affinitychromatography, ion-exchange chromatography, hydrophobic chromatographyand chromatography utilizing antibody-bound carrier. A number of methodsfor producing a substance by genetic engineering process are known andMCAF can be produced by combining the known processes.

Alternatively, cells which intrinsically produce MCAF or which produceMCAF upon some stimulation, such as monocytes, fibroblasts, endothelialcells, keratinocytes, smooth muscle cells, astrocytes, or cell linessuch as THP-1 (myelomonocyte) and U-105MG (glioma) are cultured so as tomake the cells produce MCAF with or without appropriate stimulation; andMCAF may then be purified by the above-mentioned purification method.MCAF-producing cells and the conditions for stimulating the cells aredescribed in the above-mentioned References 4) and 5).

Human MCAF having the amino acid sequence shown in SEQ ID NO. 1 in theSequence Listing is commercially available (see Example 1 below), andsuch a commercially available MCAF may suitably be employed in thepresent invention.

The term "wound" herein means damages of tissues including skin andmucous membrane, and includes skin ulcers. Skin ulcers include traumaticulcers caused by burn, trauma or surgery; circulatory disorder ulcerssuch as decubitus, cnemial ulcers and the like; and basic disorderulcers such as skin diseases, and endogenous (digestive fluid) ulcers.In the examples, the effectiveness of the present invention is clearlyshown using a skin wound model of rabbits. From the prominenteffectiveness in curing wounds shown in the examples, the therapeuticeffect of MCAF for curing skin ulcers, especially traumatic ulcers iseasily expected.

To treat the wounds, which is an object of the present invention, acomposition comprising MCAF or a variant thereof having amonocyte-attracting property or a derivative of MCAF or a variantthereof in a pharmaceutically acceptable carrier is administered to thebody. The wounds to which the composition is administered are, asmentioned above, damages of tissues including skin and mucous membrane,and include skin ulcers such as traumatic ulcers caused by burn, traumaor surgery; circulatory disorder ulcers such as decubitus, cnemialulcers and the like; and basic disorder ulcers such as skin diseases,and endogenous (digestive fluid) ulcers.

Other components to be blended may be water, organic solvents or othergeneral pharmaceutically acceptable additives. Needless to say, however,the object of the present invention may also be attained without anadditive. Examples of the pharmaceutically acceptable additives includecollagen, heparin, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinylpolymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodiumalginate, hyaluronic acid, water-soluble dextran, sodium carboxymethylstarch, pectin, methyl cellulose, ethyl cellulose, xanthane gum, gumarabi, casein, gelatin, agar, polyethylene glycol, diglycerin, glycerin,propylene glycol, polyethylene glycol, vaseline, paraffin, stearylalcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol,lactose and pharmaceutically acceptable surfactants. The additives maybe appropriately selected depending on the formulation of thetherapeutic agent of the present invention from the above-mentionedadditives and combinations thereof, but, needless to say, the additivesare not restricted to those mentioned above.

According to the present invention, other drugs, biological formulationsand synthetic pharmaceuticals may be simultaneously or sequentiallyadministered together with MCAF. The other drugs may be those havinganti-inflammatory activities, peripheral circulation-improvingactivities, thrombus formation-inhibiting activities, tissue-repairingactivities; those known to have therapeutic effects for treatingalimentary canal ulcers; heparin; or therapeutic agents for treatingwounds which reinforce or supplement the effects of the presentinvention.

Although the method of administration is not restricted, in view of theobject of the present invention, topical administration is preferred.Formulations for topical administration include external formulations,suppositories and injection solutions. When treating alimentary canalulcers, oral administration can be appropriately selected. Externalformulations include application formulations such as ointments, gels,creams, emulsions and lotions; pasting formulations such as tapes andpatches; sprays; and powders. Administration dose of the effectiveingredient may be selected from the range of 0.001 μg to 100 mg perwound per day, preferably 0.01 μg to 1 mg per wound per day. However,the administration dose varies depending on the size and state of thewound and so the administration dose is not restricted to the rangementioned above. When used as an external formulation, the content ofthe effective ingredient in the formulation may be 0.000001% by weightto 10% by weight, preferably 0.00001% by weight to 0.1% by weight. Thetimes of administration may be once when wounded, or one to severaltimes per day, or once per two or several days, although not restrictedthereto.

EXAMPLES

The present invention will now be described in more detail and moreconcretely by way of examples. Needless to say, however, the presentinvention is not restricted by the examples.

Example 1

Effect of MCAF on Wounds of Rabbits

1) Testing Method

As the rabbits, NZW (New Zealand White) rabbits were employed, and eachgroup consisted 4 rabbits.

After anesthetizing each rabbit with 75 mg of pentobarbital, fiveincised wounds having a length of 5 cm were formed in the back of eachrabbit. As MCAF, human MCAF prepared by genetic recombination, which iscommercially available from Pepro tech, Inc., Rocky Hill, N.J. 08553,USA was used.

The compositions described below (drug and control drugs) were topicallyinjected. 0.1 ml of the composition was injected per each injection, andtotally 1 ml of the composition was injected per wound.

(1) PBS (1 ml)

(2) PBS (1 ml)+bovine collagen (1 mg)

(3) PBS (1 ml)+bovine collagen (1 mg)+IL-8 (5 μg)

(4) PBS (1 ml)+bovine collagen (1 mg)+IL-1 (5 μg)

(5) PBS (1 ml)+bovine collagen (1 mg)+MCAF (5 μg)

Rabbits were sacrificed 7 days or 14 days after the injection. Theentire skin of the back of each rabbit was peeled off and samples in theform of a tape having a width of 1.5 cm, containing the incised wound ata right angle to the tape were prepared. The tensile strengths of thetapes were measured. The tensile strength was measured by usingAutograph Tension Tester commercially available from Shimazu Seisakusho,in accordance with the method described in Watanabe, Y. et al., Surg.Res. Comm., 10, 267-277, 1991.

2) Test Results

The results obtained 7 days after the injection are shown in Table 1 andFIG. 1, and the results obtained 14 days after the injection are shownin Table 2 and FIG. 2.

                                      TABLE 1                                     __________________________________________________________________________    Tensile Strength of Wounded Skin at 7 Days after Wounding                                      Measured Tensile Strength (grams)                            Administered Composition                                                                       Rabbit 1                                                                           Rabbit 2                                                                           Rabbit 3                                                                           Rabbit 4                                                                           Average                                                                            SD                                  __________________________________________________________________________    PBS              205  250  130  405  248  116                                 PBS + bovine collagen                                                                          165  220  105  440  233  146                                 PBS + bovine collagen + IL-8                                                                   345  485  165  760  439  251                                 PBS + bovine collagen + IL-1                                                                   590  590  250  565  498  166                                 PBS + bovine collagen + MCAF                                                                   685  1225 320  1395 906  494                                 __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Tensile Strength of Wounded Skin at 14 Days after Wounding                                     Measured Tensile Strength (grams)                            Administered Composition                                                                       Rabbit 5                                                                           Rabbit 6                                                                           Rabbit 7                                                                           Rabbit 8                                                                           Average                                                                            SD                                  __________________________________________________________________________    PBS              2250 2300 1060 1480 1772  605                                PBS + bovine collagen                                                                          1170 2900 2560  704 1584  945                                PBS + bovine collagen + IL-8                                                                   3275 1925 1470 1428 2025  864                                PBS + bovine collagen + IL-1                                                                   3310 3225 2350 1960 2711  663                                PBS + bovine collagen + MCAF                                                                   4370 4000 4650 1900 3730 1249                                __________________________________________________________________________

Table 1 shows the measured tensile strengths of the skin samples 7 daysafter the injection and Table 2 shows the measured tensile strengths ofthe skin samples 7 days after the injection in terms of grams. Each ofthe 4 values shown as the actually measured values correspond to each ofthe 4 rabbits from the left column. The average and standard deviation(SD) are shown in the right columns.

From Table 1, FIG. 1, Table 2 and FIG. 2, an increase in the tensilestrength is clearly shown in those samples to which MCAF wasadministered. Thus, it is concluded that MCAF is effective as atherapeutic agent for treating wounds. A statistical analysis (Student'st test) was performed on Table 1. The group to which MCAF wasadministered, that is, the group to which composition (5) wasadministered, showed significant difference with a significanceprobability of not more than 0.05 with respect to the group to which PBSalone was administered, that is, the group to which composition (1) wasadministered, and with respect to the group to which collagen wasadministered, that is, the group to which composition (2) wasadministered. Although no significant differences were observed amongother values and among the values in Table 2 in this statisticalanalysis because of the limited number of the animals, by preciselycomparing the measured value of each rabbit in Tables 1 and 2, anincrease in the tensile strength is clearly observed in the group towhich MCAF was administered.

Example 2

The therapeutic effects of MCAF for skin wounds were compared with thoseof other growth factors. Further, dose dependence of the therapeuticeffects of MCAF was examined.

1) Testing Method

The following experiments were carried out in accordance with the methodin Example 1 in principle.

As the MCAF, one obtained from human fibroblast cells stimulated bypolyI:polyC 50 mg/l which was purified by hydrophobic chromatography,cation-exchange chromatography and reverse phase HPLC, was employed.This MCAF exhibited the same physiological actions as the MCAFcommercially available from Pepro tech, Inc.

NZW female rabbits (body weight: 2.5-3.5 kg) were used. Anesthesia wasperformed by administering 25 mg/kg of pentobarbital. The length of theincised wounds in the skin of the back was 6 cm. The wounds were closedby surgical staples at 1.5 cm intervals. MCAF or other growth factorswere formulated into phosphate buffer containing 10 mg/ml of emulsifiedcollagen (Zyderm Collagen Corp., Palo Alto, Calif.). When closing thewounds by staples, 0.5 ml of the above-mentioned emulsion wasadministered per each wound. Seven days after the administration,rabbits were sacrificed and skin samples in the form of a tape having awidth of 1.5 cm, each of which contains the wound at its center at rightangles to the tape, were prepared. Two micrograms of EGF (recombinanthuman epidermal growth factor) commercially available from BectonDickinson, 2 μg of EGF (recombinant human basic fibroblast growthfactor) commercially available from Genzoyme, 2 μg of TGF-α (recombinanthuman transforming growth factor-α) commercially available from BectonDickinson or 2 μg of TGF-β (human transforming growth factor)commercially available from Becton Dickinson was applied to each wound.

In the test for examining the dose dependence of the therapeutic effectof MCAF, the administration dose of MCAF per wound was 0.2, 1.0 or 5.0μg.

In all cases, the skin samples were tested for their tensile strengthsin the same manner as in Example 1 and the results are shown in terms ofgm (gram). The results are shown in FIGS. 3 and 4. The results are shownin terms of average ±SD (standard deviation) and statistical analysis(Student's t test) was performed.

In addition to the tensile strength test, each skin sample was stainedwith hematoxylin-eosin after being fixed by formalin, and granulation atthe sutured regions was observed.

2) Results

Comparison among the effects by MCAF and by other growth factors areshown in FIG. 3. The rupture strength was 267.5±59.6 grams when PBSalone was administered, 385.4±189.9 grams when bFGF was administered,and 645.0 ±188.8 grams when MCAF was administered. The results of thegroup to which MCAF was administered were significant with respect tothe group to which PBS alone was administered with p<0.05. Further, MCAFwas more effective than bFGF. By microscopic examination, nopathological abnormalities were observed in any case.

The dose dependence of the effects of MCAF is shown in FIG. 4. Therupture strength was 275.1±90.5 grams when PBS alone was administered,471.0±193.3 grams when 0.2 μg of MCAF was administered, 504.6±23.7 gramswhen 1.0 μg of MCAF was administered and 644.0±128.2 grams when 5.0 μgof MCAF was administered. Differences between pathological observationsdepending on the dose of MCAF were not observed.

INDUSTRIAL AVAILABILITY

As described above, the therapeutic agent for treating wounds accordingto the present invention exhibits excellent therapeutic effect forwounds. Therefore, it is expected that the therapeutic agent fortreating wounds according to the present invention will very muchcontribute to therapy of wounds in the medical field. ##STR1##

We claim:
 1. A method of treating wounds comprising administering to apatient in need thereof an effective amount of a therapeutic agenthaving as an active ingredient a compound selected from the groupconsisting of monocyte chemotactic and activating factor, a variantthereof having monocyte attracting properties, a derivative of monocytechemotactic and activating factor and a variant of said derivative.
 2. Amethod of claim 1 wherein said therapeutic agent is administered in aform selected from the group consisting of external formulations,suppositories, injections and orally.
 3. A method of claim 1 whereinsaid active ingredient is administered in a range of 0.001 μg to 100 mgper wound per day.
 4. A method of claim 1 wherein said therapeutic agentis administered one to several times per day.
 5. A method of claim 1wherein said wound is selected from the group consisting of damagedtissues; skin ulcers caused by burns, trauma or surgery; circulatorydisorder ulcers; skin diseases; and digestive fluid ulcers.
 6. Themethod of claim 1, wherein said variant thereof is obtained by thedeletion, substitution, or addition of amino acid residues of MCAF. 7.The method of claim 1, wherein said derivative thereof is obtained bybiochemical modification of MCAF, wherein said modification is selectedfrom the group consisting of chemical linking with polyethylene glycol,phosphate group attachment, sulfate group attachment, peptidasetreatment, treatment with a sugar chain-modifying enzyme, and treatmentwith a sugar attachment enzyme.
 8. The method of claim 1, wherein saidvariant thereof is selected from the group consisting of RANTES, MCP-2,MCP-3, LD78, ACT2, 1-309, JE, MIP-1α, MIP-1β, and TCA.
 9. The method ofclaim 6, wherein said variant is obtained by deletion or addition ofamino acid residues from the amino terminal end of MCAF.
 10. The methodof claim 6, wherein said variant is obtained by deletion or addition ofamino acid residues from the carboxy terminal end of MCAF.